The initial observations of the “hybridization” process, i.e., the ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction, by Marmur and Lane, Proc. Nat. Acad. Sci., U.S.A. 46, 453 (1960) and Doty, et al., Proc. Nat. Acad. Sci., U.S.A. 46, 461 (1960), have been followed by the refinement of this process into an essential tool of modern biology. Initial hybridization studies, such as those performed by Hayashi, et al., Proc. Nat. Acad. Sci., U.S.A. 50, 664 (1963), were formed in solution. Further development led to the immobilization of the target DNA or RNA on solid supports. With the discovery of specific restriction endonucleases by Smith and Wilcox, J. Mol. Biol. 51, 379 (1970), it became possible to isolate discrete fragments of DNA. Utilization of immobilization techniques, such as those described by Southern, J. Mol. Biol. 98, 503 (1975), in combination with restriction enzymes, has allowed for the identification by hybridization of singly copy genes among a mass of fractionated, genomic DNA.
Khorana, Kleppe and Molineaux recognized that hybridization and primer extension with a polymerase could be utilized to amplify nucleic acid (Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana H G, “Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases.” J Mol Biol 56:341-61, 1971). Later, a thermostable polymerase, Taq DNA polymerase, was employed in this process, now known as PCR (Innis M A, Myambo K B, Gelfand D H, Brow M A, “DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA.” Proc Natl Acad Sci USA 85:9436-40, 1988). Taq polymerase is a thermostable enzyme which works efficiently at 70-75° C. The ability to catalyze DNA synthesis at elevated temperature makes Taq polymerase useful because this permits hybridization at higher temperatures, giving PCR greater specificity for the target to be amplified. Taq polymerase is also useful for sequencing templates which have extensive secondary structures at 37° C. (the standard temperature used for Klenow and Sequenase™ reactions).
Typically, in carrying out PCR one utilizes a high temperature (e.g., 95° C.) to separate the DNA and a lower temperature (e.g., 65° C.) to anneal and extend. In some cases, a different temperature is used for the extension, causing the reaction to require changing the temperature three times per cycle. The temperature changes are usually accomplished with a specialized device known as a “thermocycler” (commercially available from a number of companies, including F. Hoffmann-La Roche Ltd., Roche Molecular Systems, Inc., Nutley, N.J., USA and The Perkin-Elmer Corporation, Wellesely, Mass., USA). These devices are expensive and constrain the user to relatively large reaction volumes.
What is needed is a method for changing the temperature of reactions that is simple and inexpensive. Importantly, the method should be amenable to a variety of reaction types and reaction volumes.